Epidemiology of Infantile Visceral Leishmaniasis in Western Algerian And The Convenience of Serum For The Disease Diagnosis by PCR and Immunochromatography

Epidemiological situation of infantile visceral leishmaniasis (IVL), which is a public health problem in Algeria, is almost unknown in the cities of Western part of the country. The aim of this study was to analyze the epidemiological, clinical, biological, therapeutic, and evolutionary aspects of IVL in Western Algeria, to evaluate the performance of the immunochromatography as a rapid diagnostic test of the disease, and to propose a diagnosis approach by real-time polymerase chain reaction (RT-PCR) assay from the serum. This prospective study was performed on 63 suspicious cases of visceral leishmaniasis collected from the infectious diseases department at the Pediatric Hospital of Oran from January 2012 to July 2017. For each patient, the epidemiological parameters, and the clinical and biological data were collected. Bone marrow and blood samples were drawn from all cases. Bone marrow was performed to research amastigote forms of Leishmania and to identify the species by PCR-sequencing. Blood samples were used to detect anti-Leishmania antibodies as well as parasite DNA. Patients from the Western regions were mostly from rural areas. Sensitivity of RT-PCR from the bone marrow and from serum was 95.45% and 94.44%, respectively. The immunochromatography allowed the disease’s diagnosis for 11 cases whose myelogram did not confirm the presence of the amastigote forms of Leishmania. Immunochromatography was revealed to be a good technique for disease diagnosis regarding the strongly evocative clinical signs. The results also suggest the interest of the RT-PCR assay from patient serum as a non-invasive sample, in the detection of parasite DNA.

For each patient included in the study, the epidemiological parameters (age, sex, geographical origin, etc.), the clinical and biological data, were collected from the medical record. Bone marrow and blood samples were drawn from all cases. The anonymity of the patients and the confidentiality of their information were respected.
Bone marrow, collected at the level of the iliac crest, was spread in thin smear, on slides, fixed with methanol or the May Grünwald, and colored with Giemsa for the research of Leishmania's amastigote forms. After reading, the slides were kept at room temperature.
The blood samples drawn in dry tube were centrifuged at 1509 xg for 5 min. After separation, 20 µl of sera were placed on the absorbent part on the strip of the rapid test by immunochromatography for the detection of anti-Leishmania antibodies. The remaining sera were later aliquoted into Eppendorf tubes, and stored at-20 C.
To evaluate its specificity, the immunochromatography was carried out on 27 serum samples of patients with other pathologies (tuberculosis, malaria, toxoplasmosis, brucellosis, hepatitis B, cutaneous leishmaniasis, acquired immunodeficiency syndrome (AIDS), and polyarthritis), and 10 serum samples from negative controls.
A total of 22 bone marrow smears from confirmed cases were scraped using a sterile scalpel. The scraping powder of each of these smears was placed in a previously coded sterile Eppendorf tube. To evaluate its specificity, the bone marrows of 9 cases with other pathologies as well as that of a negative control case were introduced in the study.
The sera and powders of the conserved bone marrows were intended for molecular study by RT-PCR and PCR-sequencing.
The research protocol was approved by the Ethics Committee of University Hospital of Oran (EHU).

Statistical analysis
In this descriptive study, all parameters have been analyzed by the Epi Info software version 6.04d f. The average and standard deviation were calculated for the quantitative variables and the percentage for the qualitative variables.
Sensitivity, specificity, negative and positive predictive values were estimated with confidence intervals at 95%.
The Youden Index (j) and the coefficient of Yule (Q) were introduced for the first time in the evaluation of the rapid test's performance by immunochromatography (rk39).

Results
A total of 39 patients were retained in this study.
The others (n=24) were excluded because other

Discussion
In the Maghreb countries, IVL is predominant in young children in which 95% of cases are less than 5 years old, realizing the infantile Mediterranean Kala-azar (6). The disease in this age group has already been registered in Algeria (7,8). The disease occurrence is explained by a beam of arguments among which the immaturity of the child's immune system and the sandfly's most marked affinity for this latter (9).
The appearance age of the disease corresponds to the period in which the infant loses maternal antibodies (7-15 months) and becomes sensitive to pathogens (10). However, cases were indicated in young adolescents aged from 13 to 14 years old (11). These results support those in our series.
A slight male dominance is noted in the majority of series (12), which reinforces our results.
But according to the synthesis made on the VL by Infantum from July to October, which could also explain the appearance of the first symptoms The case distribution of our study throughout the year and their late hospitalization has led to serious complications and even death.
As clinical signs, the majority of the patients had a splenomegaly, a skin-mucous pallor, and a fever that resisted antibiotic treatment. The triad: paler-splenomegaly-fever, signing in favor of an IVL, was observed in 71.79% of cases, and hepatomegaly was present in in 64.10% of cases.
These results are close to those found in other studies (Table 4)    The results of this study suggest the benefit of a diagnosis by RT-PCR that can be made from a non-invasive sample; such as the patient's serum.
The use of RT-PCR from bone marrow is recommended for highly suspected cases, with negative results. In front of evocative bio-clinical signs, the positivity of serology by immunochromatography allows a very strong presumption.
Obtaining the specific profile by a confirmation technique such as western blotting may help to remove doubts about false negatives.